Eukaryotic cells have a highly conserved enzymatic system for the ligation of ubiquitin (Ub) to cellular proteins. Polypeptides distinct from but related to Ub, called Ub-like proteins or Ubls, can also be ligated to other proteins. Ligation to each Ubl has unique mechanistic and functional consequences. SUMO (Smt3 in yeast) is a highly divergent Ubl, and the SUMO ligation system has many crucial functions, including important contributions to human health and biology. Both Ub and SUMO attachment to proteins can be rapidly reversed by specialized proteases. While analyzing the deubiquitinating enzyme (Dub) family in the yeast Saccharomyces cerevisiae, a novel family of SUMO-specific cysteine proteases unrelated in primary sequence to the Dubs was discovered. These Ubl-specific proteases (Ulps) have important functions in cell cycle progression, chromosome stability, and growth. The long-term objective of the project is to gain a molecular understanding of the physiological and mechanistic roles played by Dubs and by Ulps. In this renewal application, the proposed experiments are concentrated on SUMO modification ("sumoylation") in yeast and on the contributions of the two yeast desumoylating enzymes, Ulpl and Ulp2/Smt4, to the function of the SUMO system. The specific aims are to: 1. Identify SUMO-conjugated proteins in yeast, especially essential ones, by an array of biochemical, genetic, and proteomic approaches. 2. Identify essential physiological processes in yeast that depend on SUMO and determine the mechanistic contributions of the desumoylating enzymes to these processes. 3. Verify the sumoylation of identified substrates, locate their sites of SUMO attachment, and analyze the consequences of specific SUMO-protein modifications. 4. Determine the molecular basis for the differences in specificity and activiity between the Ulpl and U|p2 desumoylating enzymes.